Voltage-dependent calcium channels (VDCCs) control the gateways of calcium influx via the plasma membrane and thereby play essential roles in numerous biological activities such as synaptic transmission, muscle contraction, gene expression, hormone secretion, cell motility and development. Structurally, VDCCs comprise a pore-forming β subunit and as many as three auxiliary subunits: β2δ, β and γ. The auxiliary β and α2δ subunits serve as positive regulators of calcium current by promoting surface expression of the α1 subunit, enhancing voltage-dependent activation and increasing channel open probability. From recent work as set forth in this present disclosure, we have expanded the knowledge of the functions of the γ proteins as calcium channel auxiliary subunits, particularly in the context of the regulation of calcium channels. We also describe a further understanding of the mechanistic details.
The calcium channel γ family is a subgroup of tetraspanin proteins, which have four transmembrane segments and intracellular N- and C-termini. Among the eight γ subunits that have been identified, γ1 was found in the calcium channel complex in skeletal muscles. Using γ1-null mouse as an experimental model, γ1 has been shown to accelerate voltage-dependent inactivation and reduce current density of the Cav1.1 channel in the skeletal muscles. Thus the function of the γ1 subunit seems to be a negative regulator of the high voltage-activated (HVA) calcium current. There is indication that γ1 interacts with the α1.1 subunit through the first half of the γ molecule.
Despite the observation of the modulation or inhibition of HVA calcium currents by γ2, γ3, γ4 and γ7 in heterologous expression systems, certain attempts to demonstrate the influence of several γ subunits on HVA calcium current under physiological contexts (such as in neurons) have not been successful. Moreover, four of the eight subunits (γ2, γ3, γ4 and γ8) that contain a PDZ-binding motif in their C-termini have been recently found to act primarily as regulators of AMPA receptors and collectively named the transmembrane AMPA receptor regulatory proteins (TARPs). The gamma5 (γ5) and gamma7 (γ7) subunits have been proposed to act as type II TARPs that modulate glutamate receptor channels. Taken together, the biological roles of γ2, γ3, γ4, γ5, γ7 and γ8 as auxiliary subunits of calcium channels remain a subject of debate.
In contrast, γ6, which is structurally the closest homologue of γ1, is the only other γ subunit that seems to conform to the classical definition of a calcium channel subunit. In a heterologous expression system, γ6 has been shown to robustly inhibit the calcium current of Cav3.1, a low voltage-activated (LVA) channel. In International Publication No. WO/2007/041360 and Patent Application Publication No. US 20070213267, we demonstrated that γ6 not only associates with the α1 subunit of the Cav3.1 channel, or the α3.1 subunit, but also reduces LVA current density. We also identified a GxxxA motif in the first transmembrane domain (TM1) of γ6 as critical for inhibiting the Cav3.1 current.
There is still a need in the art, however, for additional innovations and improvements towards the ability to generate relatively short peptides which are active in regulating calcium channel function. Moreover, the earlier characterization of the structure of useful peptides is further advanced by the present disclosure. We identify structural features of peptides, particularly short peptides capable of demonstrating significant activity levels in affecting calcium channel function, which facilitate the generation of compositions and methods including pharmacologic agents and therapeutic applications. Embodiments of the present invention therefore address such need.